Baculoviruses are large DNA viruses that are virulent pathogens of insects and they serve as important models for studies of virus-host interactions. In addition, the baculovirus AcMNPV has been developed as a transduction vector for mammalian cell expression, with important applications in areas such as high throughput screening and the potential for use in human gene therapy. Viral entry by AcMNPV budded virions (BV) is mediated by the major envelope glycoprotein, GP64. The receptor for GP64 is not known. GP64 is also necessary for efficient virion budding and progeny virus production. In natural hosts of AcMNPV, the GP64 protein also plays a critical role in the initial phase of transmission in the insect. GP64 is targeted to basal membranes in polarized midgut epithelial cells and this targeting appears to direct budding and transmission of infection into the insect hemocoel. In the proposed studies, our work will be concentrated in three specific areas: 1) Viral receptor binding; 2) Envelope protein targeting in insect polarized midgut epithelial cells; and 3) Virion assembly and budding. Studies of viral receptor binding will focus on identification of the GP64 receptor binding domain and the host cell receptor. We will also identify the GP64 midgut targeting signal and interacting proteins involved in this process. Because GP64 is critical for efficient virion budding, we will identify the GP64 budding domain as well as viral and/or cellular proteins that interact with GP64 during assembly and budding. For these studies, we will use an engineered cell line expressing GP64, and a powerful genetic system that we recently developed, to replace wild type gp64 in the viral genome with modified forms of gp64. Using these powerful genetic tools with a range of functional assays, we will examine GP64 function in the context of budded virions and the viral infection cycle. These studies will address important and central questions, advancing our understanding of baculovirus interactions with host receptors, virus movement through insect polarized midgut epithelial cells, and the mechanism of virus budding. In addition, results of many of these studies will be directly applicable to new and exciting biotechnological applications of baculoviruses in mammalian cells. ? ?

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
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Special Emphasis Panel (ZRG1-VB (01))
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Cassetti, Cristina
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Boyce Thompson Institute for Plant Research
United States
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Chen, Yun-Ru; Zhong, Silin; Fei, Zhangjun et al. (2013) The transcriptome of the baculovirus Autographa californica multiple nucleopolyhedrovirus in Trichoplusia ni cells. J Virol 87:6391-405
Dong, Sicong; Blissard, Gary W (2012) Functional analysis of the Autographa californica multiple nucleopolyhedrovirus GP64 terminal fusion loops and interactions with membranes. J Virol 86:9617-28
Li, Zhaofei; Blissard, Gary W (2012) Cellular VPS4 is required for efficient entry and egress of budded virions of Autographa californica multiple nucleopolyhedrovirus. J Virol 86:459-72
Li, Zhaofei; Blissard, Gary W (2011) Autographa californica multiple nucleopolyhedrovirus GP64 protein: roles of histidine residues in triggering membrane fusion and fusion pore expansion. J Virol 85:12492-504
Hashimoto, Yoshifumi; Zhang, Sheng; Blissard, Gary W (2010) Ao38, a new cell line from eggs of the black witch moth, Ascalapha odorata (Lepidoptera: Noctuidae), is permissive for AcMNPV infection and produces high levels of recombinant proteins. BMC Biotechnol 10:50
Li, Zhaofei; Blissard, Gary W (2010) Baculovirus GP64 disulfide bonds: the intermolecular disulfide bond of Autographa californica multicapsid nucleopolyhedrovirus GP64 is not essential for membrane fusion and virion budding. J Virol 84:8584-95
Li, Zhaofei; Blissard, Gary W (2009) The pre-transmembrane domain of the Autographa californica multicapsid nucleopolyhedrovirus GP64 protein is critical for membrane fusion and virus infectivity. J Virol 83:10993-1004
Li, Zhaofei; Blissard, Gary W (2009) The Autographa californica multicapsid nucleopolyhedrovirus GP64 protein: analysis of transmembrane domain length and sequence requirements. J Virol 83:4447-61
Li, Zhaofei; Blissard, Gary W (2008) Functional analysis of the transmembrane (TM) domain of the Autographa californica multicapsid nucleopolyhedrovirus GP64 protein: substitution of heterologous TM domains. J Virol 82:3329-41
Zhou, Jian; Blissard, Gary W (2008) Display of heterologous proteins on gp64null baculovirus virions and enhanced budding mediated by a vesicular stomatitis virus G-stem construct. J Virol 82:1368-77

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